The objective of this proposal is to develop improved methods for inducible genetic marking, mapping and manipulation of specific brain regions or neuronal subpopulations in the mammalian brain. In Aim I, methods for isolating brain region-restricted and cell type-specific genes utilizing laser-capture microdissection together with DNA microarray analysis will be developed and optimized. These experiments will focus on identifying markers for limbic system structures, including the amygdala, BNST, hypothalamus and lateral septum. In Aim II, the utility and efficacy of different genetically encoded primary axonal markers will be compared, and genetically encoded, inducible anterograde and retrograde trans-neuronal tracers will be developed and tested in vivo. Proof-of-principle will first be established in the PNS using a well-defined model system, and then extended to the CNS. Aim III encompasses the development and in vivo testing of two alternative combinatorial, positive coincidence-detection systems for achieving region- or cell subtype-specific control of heterologous gene expression (e.g., neuronal tracers or silencers), without having to identify specific transcriptional enhancer elements for such regions or cells. One method is based on inducible site-specific DNA recombination. The other method is based on a "two-hybrid" system for inducible transcriptional activation. In both cases, expression of the reporter/tracer gene is induced only in cell populations in which the expression of two different "co-driver" genes overlaps. This "Venn diagram" approach allows different pairwise combinations of driver genes to be used to express reporter or tracer genes in only a restricted subset of the regions in which each individual co-driver gene is expressed. These methods will initially be developed, tested and optimized using highly specific genes with known overlapping patterns of expression in specific subsets of pain-sensing primary sensory neurons. In Aim IV, based on the outcome of Aim III, either the recombination-based or two-hybrid system will be selected for extension to the CNS, using an overlapping pair(s) of limbic system-specific genes identified in Aim I. In addition, the recombination-based system will be extended to permit activity-dependent trans-neuronal tracing of neurons expressing a specific marker gene. The combination of new limbic system-specific molecular markers and genetically encoded tract-tracing and neuronal ablation methods should improve our understanding of the functional neuroanatomy of emotion and affective disorders such as anxiety and depression.